The isolation of rat alveolar type II cells: a simplified approach using Percoll density centrifugation

Skillrud, David M.; Martin, William J.

doi:10.1007/bf02715652

Cited by 19 publications

(9 citation statements)

References 23 publications

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“…After 90 min, the nonadherent cells were removed, centrifuged (250 g for 10 min), and resuspended in solution I. For further purification, the suspension was applied to a Percoll gradient established by centrifuging the combination of Percoll with phosphate-buffered saline (25 mmol/ liter NaH2PO4, 300 mmol/liter NaCl, pH 7.4), in a 6:7 (vol/vol) ratio for 10 min at 20,000 g (12). Cells were counted in a standard hemacytometer.…”

Section: Methodsmentioning

confidence: 99%

Tumor necrosis factor-alpha-induced inhibition of phosphatidylcholine synthesis by human type II pneumocytes is partially mediated by prostaglandins.

Arias-Dı́az

Vara²,

Garcı́a³

et al. 1994

J. Clin. Invest.

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Section: Methodsmentioning

confidence: 99%

Tumor necrosis factor-alpha-induced inhibition of phosphatidylcholine synthesis by human type II pneumocytes is partially mediated by prostaglandins.

Arias-Dı́az

Vara²,

Garcı́a³

et al. 1994

J. Clin. Invest.

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“…After 90 min, the nonadherent cells were removed, centrifuged (250 ϫ g for 10 min), and resuspended in solution I. For further purification, the suspension was applied to a Percoll gradient by centrifuging the combination of Percoll with phosphate-buffered saline (25 mM NaH 2 PO 4 , 300 mM NaCl, pH 7.4) in a 6:7 (v/v) ratio for 10 min at 20,000 ϫ g (27). Cells were counted in a standard hemacytometer.…”

Section: Type II Pneumocyte Isolation Proceduresmentioning

confidence: 99%

Glucagon-like Peptide-1(7–36) Amide Stimulates Surfactant Secretion in Human Type II Pneumocytes

Vara

Arias-Dı́az²,

Garcı́a³

et al. 2001

Am J Respir Crit Care Med

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To determine the influence of glucagon-like peptides on the secretion of human pulmonary surfactant, we used human type II pneumocytes. In these cells, GLP-1(7-36) amide and exendin-4 stimulated phosphatidylcholine secretion (PC) and cAMP formation in a concentration-dependent manner; these effects were reversed by exendin(9-39). No changes were observed with other related peptides. The mechanism by which GLP-1(7-36) amide exerts its stimulatory effect was investigated with various agents that are well known to be stimulators or inhibitors of PC secretion. Thus, 8-bromo-cAMP increased and both Rp-cAMPS and H-89, the latter an inhibitor of protein kinase A (PKA), reduced pulmonary surfactant secretion in type II pneumocytes. Also, GLP-1(7-36) amide and TPA exerted additive effects in stimulating PC secretion, and Calph C, a potent inhibitor of protein kinase C (PKC), blocked most of the effect of GLP-1(7-36) amide. By contrast, both the calcium ionophore A23187 and GLP-1(7-36) amide had additive effects in increasing PC secretion, and the specific inhibitor of Ca(2+)-calmodulin-dependent protein kinase (Ca-CM-PK), KN-62, inhibited the effect of A23187 but did not alter the stimulatory action of GLP-1(7-36) amide. Our findings suggest that both PKA and PKC are involved in the stimulatory effects of GLP-1(7-36) amide on PC secretion, whereas this peptide has no effect on PC secretion through a Ca-CM-PK mechanism.

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“…These include unit gravity separation or centrifugal elutriation (Brown et al, 1984;Wilson et al, 1986;Massey et al, 1987), centrifugation of the crude cell mixture on a discontinuous gradient of percoll (Skilrud and Martin, 1984;Richards et al, 1987;Augustin-Voss et al, 1989), ficoll (Kikkawa and Yoneda, 1974;Fisher and Furia, 1977;Kikkawa et al, 1975;Finkelstein and Shapiro, 1982;Pfleger, 1977), or metrizamide (Dobbs et al, 1980;Goodman and Crandall, 1982;Cheek et al, 1989;Sikpi et al, 1986;Robinson et al•, 1984), and removal of blood-borne cells adhering to leukocyte-common antigen (Weller and Karnovsky, 1986a;Banks et al, 1991) or IgG (Dobbs et al, 1986;Banks et al, 1991) coated on a hydrophobic surface. The IgG panning method may be the least time-consuming.…”

Section: Primary Culture Of Alveolar Pneumocytesmentioning

confidence: 99%

Studies on the mechanisms of active ion fluxes across alveolar epithelial cell monolayers

Kim

Suh

Lubman

et al. 1992

Journal of Tissue Culture Methods

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SUMMARY: To investigate the celt physiologic and biological properties of the alveolar epithelium, we studied rat alveolar epithelial cell monolayers grown on permeable supports in primary culture. Type II alveolar epithelial cells were disaggregated using elastase, and partially purified on a discontinuous metrizamide gradient. These isolated cells were plated onto tissue culture-treated Nuclepore membrane filters at 1.5 )< 106 cells/cm 2 and maintained in a humidified incubator (5% CO2 in air, 37 ° C). After 2 days in culture, the bathing media on both sides of the cell monolayers were changed to fresh culture medium, thus removing nonadherent cells (mostly lcukocytes). These monolayers exhibit a high transmonolayer resistance (>2000 ~-cm 2) and actively transport ions. Radionuclide flux studies indicate that Na + is the predominant ionic species absorbed actively under baseline conditions, accounting for about 80% of the total active ion transport. CI-seems to be passively transported across the epithelium. However, when the epithelium is exposed to a beta-agonist (terbutaline), active absorption of Na + is increased ar, d active absorption of C1-occurs. Ahhough it is clear that both active Na + and CI-transport are dependent on Na+/K+-ATPase activity, and that Na + enters cells predominantly through channels, the specific mechanisms by which CI-enters and exits the alveolar epithelial cells remain unclear. The stimulated reabsorption of Na + and CImay be important in helping to remove excess fluid from alveolar air spaces in the lung.

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The isolation of rat alveolar type II cells: a simplified approach using Percoll density centrifugation

Cited by 19 publications

References 23 publications

Tumor necrosis factor-alpha-induced inhibition of phosphatidylcholine synthesis by human type II pneumocytes is partially mediated by prostaglandins.

Tumor necrosis factor-alpha-induced inhibition of phosphatidylcholine synthesis by human type II pneumocytes is partially mediated by prostaglandins.

Glucagon-like Peptide-1(7–36) Amide Stimulates Surfactant Secretion in Human Type II Pneumocytes

Studies on the mechanisms of active ion fluxes across alveolar epithelial cell monolayers

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